Thermo Fisher Scientific ProQuest™ Two-Hybrid System User guide

Brand: Thermo Fisher Scientific

Model: ProQuest™ Two-Hybrid System

Type: User guide

For Research Use Only. Not for use in diagnostic procedures.

ProQuest™ Two-Hybrid System

USER GUIDE

A sensitive method for detecting protein-protein interactions

Catalog Number PQ1000101

Publication Number MAN0000537

Revision B.0

Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USA

Revision history:MAN0000537 B.0 (English)

Revision Date Description

B.0 1 December 2023 Removed Cat. No. PQ10002-01. Removed references. Updated Gateway™ Technology Open Architecture Policy.

A.0 24 October 2005 New document for ProQuest™ Two-Hybrid System.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE

LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR

ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product,

you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed.

Contents

■CHAPTER1Productinformation .................................................. 8

Contents ....................................................................... 8

Contents and storage ....................................................... 8

Shipping and storage ........................................................ 8

ProQuest™ Two-Hybrid system reagents ....................................... 9

GenotypeMaV203 .......................................................... 9

Accessory products ............................................................ 10

Introduction ............................................................... 10

MaV203 competentcells ................................................... 10

Accessory products ........................................................ 10

3-Aminotriazole ........................................................... 11

Zymolyase™............................................................... 11

Cycloheximide ............................................................ 11

Yeastmedia ............................................................... 12

■CHAPTER2General introduction ................................................ 13

Overview ...................................................................... 13

ProQuest™ Two-Hybrid System .............................................. 13

Supportedapplications ..................................................... 13

Advantages of the ProQuest™ Two-Hybrid system ............................. 13

Systemcomponents ....................................................... 13

Gateway™ Technology ...................................................... 14

Purpose of thismanual ..................................................... 14

Important ................................................................. 14

General description of the Two-Hybrid system ..................................... 15

Two hybrid proteins ........................................................ 15

Reporters under control of UAS ............................................. 16

Interaction drives expression of reporters ..................................... 16

Evaluating reporter gene expression ......................................... 17

Screening Two-Hybrid libraries .............................................. 17

Falsepositives ............................................................ 17

Advanced™ Two-Hybrid systems ............................................ 18

ProQuest™ Two-Hybrid system .................................................. 19

Verifying Two-Hybrid interaction ............................................. 19

Forward Two-Hybrid library screen ........................................... 19

Cloning vectors ............................................................ 19

ProQuest™ Two-Hybrid System User Guide 3

Two-Hybrid control vectors ................................................. 20

MaV203 yeast strain ....................................................... 21

Reducing falsepositives .................................................... 21

Three reportergenes ....................................................... 21

Fourphenotypes .......................................................... 22

Low-Copy-Number vectors ................................................. 22

Gateway™compatibility .................................................... 22

Plasmidshuing .......................................................... 23

Features of pDEST™32 ..................................................... 23

Features of pDEST™22 ..................................................... 23

Features of pEXP™-AD502 .................................................. 24

Plasmidisolation .......................................................... 24

Features of yeast strainMaV203 ............................................. 24

GenotypeMaV203 ......................................................... 25

Considerations in designing a Two-Hybrid screen .................................. 26

Introduction ............................................................... 26

Transcriptional activator .................................................... 26

Proteinfamily ............................................................. 26

Expression pattern ......................................................... 26

Interaction artifacts ........................................................ 27

Conﬁrmation of interaction .................................................. 27

Applications for ProQuest™ Two-Hybrid screen .................................... 28

Supportedapplications ..................................................... 28

Verifying interaction ........................................................ 28

Forward Two-Hybrid library screen ........................................... 28

■CHAPTER3Verifying interaction ................................................. 29

Introduction ................................................................... 29

Flowchart ................................................................. 30

Methods ...................................................................... 31

Generating bait and preyplasmids ........................................... 31

Testing speciﬁc Two-Hybrid interaction ....................................... 40

■CHAPTER4Forward Two-Hybrid library screen ................................ 49

Introduction ................................................................... 49

Required reagents before starting ............................................ 49

Choosing Two-Hybrid library ................................................ 49

Bait-Speciﬁc positive interaction control ...................................... 50

Flowchart ................................................................. 50

Methods ...................................................................... 51

Testingbait ............................................................... 51

Screening forward Two-Hybrid library ........................................ 55

What to do next ........................................................... 61

Interpretation of results ..................................................... 66

Contents

4ProQuest™ Two-Hybrid System User Guide

■CHAPTER5Troubleshooting ..................................................... 69

Generating bait and preyplasmid ................................................ 69

Testing speciﬁc Interaction/ retransformationassay ................................ 70

Testingbait .................................................................... 71

Forward two hybrid screen ...................................................... 72

Isolation of preyplasmid ........................................................ 73

■APPENDIXA Gateway™ recombination reactions ............................. 74

Introduction ................................................................... 74

Recombination reactions ........................................................ 74

BP reaction ............................................................... 74

LR reaction ............................................................... 74

Characteristics of modiﬁed att sites .............................................. 75

Speciﬁcity of modiﬁed att sites .................................................. 75

Vectors in ProQuest™ system .................................................... 75

Gateway™ vectors .............................................................. 76

Selection of Gateway™ vectors .................................................. 76

ccdBgene .................................................................... 77

Propagating Gateway™ vectors .................................................. 77

■APPENDIXBRecipes ............................................................. 78

Recipedetails ................................................................. 78

SC medium and plates ..................................................... 78

LB (Luria-Bertani) medium and plates ........................................ 79

5FOA plates ............................................................... 79

3AT plates ................................................................ 79

Cycloheximide plates ...................................................... 80

10xLiAc .................................................................. 80

10xTE .................................................................... 80

1X LiAc/0.5XTE ........................................................... 80

1X LiAc/1XTE ............................................................. 80

1X LiAc/40% PEG-3350/1XTE .............................................. 80

Zbuer ................................................................... 81

■APPENDIXCSupplementary protocols ......................................... 82

Generating a cDNA library using Three-Frame ..................................... 82

Three-Frame .............................................................. 82

Advantages of the Three-Frame libraries ...................................... 82

Three reading frame adapters ............................................... 82

Reduced5’UTRs .......................................................... 83

Contents

ProQuest™ Two-Hybrid System User Guide 5

Reduced Poly-Asequences ................................................. 83

Preparation of Three-Frame libraries .......................................... 83

Library scale yeast transformation (Purchased competentCells) ..................... 84

Introduction ............................................................... 84

Library scale competent yeastcells .......................................... 84

Transformants per screen ................................................... 84

Materialsneeded .......................................................... 84

Important ................................................................. 85

Transformation procedure ................................................... 85

Preparing and transforming competent cells (LibraryScale) .......................... 87

Introduction ............................................................... 87

Important ................................................................. 87

Materialsneeded .......................................................... 87

Preparing competentcells .................................................. 88

Transforming competentcells ............................................... 88

Replica Plating/Replicacleaning ................................................. 89

Introduction ............................................................... 89

Essentialtips .............................................................. 89

Procedure for replicaplating ................................................ 90

Procedure for replicacleaning ............................................... 90

Cleaningvelvets ........................................................... 90

Alternatives to replicaPlating/Cleaning ....................................... 90

Quantitative β-Galactosidase assays in liquid cultures .............................. 91

Introduction ............................................................... 91

Note ..................................................................... 91

ONPGassay .............................................................. 91

CPRGassay .............................................................. 92

Plasmidshuing ............................................................... 94

Introduction ............................................................... 94

Procedure for plasmidshuing .............................................. 94

■APPENDIXDMaps and features of vectors .................................... 96

pDEST™32 .................................................................... 97

Map of pDEST™32 ......................................................... 97

pDEST™22 .................................................................... 98

Map of pDEST™22 ......................................................... 98

Features of vectors pDEST™32 and pDEST™22 .................................... 99

Features of vectors ........................................................ 99

pEXP™-AD502 ................................................................ 100

Map of pEXP™-AD502 ..................................................... 100

MCS of pEXP™-AD502 .................................................... 101

pEXP™32-Krev1 .............................................................. 102

Map of pEXP™32-Krev1 ................................................... 102

Contents

6ProQuest™ Two-Hybrid System User Guide

pEXP™22-RalGDS-wt, m1,m2 .................................................. 103

Maps of pEXP™22-RalGDS-wt, m1,m2 ..................................... 103

Map of pENTR™-gus .......................................................... 105

Description .............................................................. 105

Map of control vector ..................................................... 105

■APPENDIXESupplementalinformation ....................................... 106

Gateway™ Technology ......................................................... 106

■APPENDIXFSafety .............................................................. 107

Chemicalsafety .............................................................. 108

Biological hazardsafety ....................................................... 109

■Documentation and support ...................................................... 110

Customer and technical support ................................................ 110

Limited product warranty ...................................................... 110

Contents

ProQuest™ Two-Hybrid System User Guide 7

Product information

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix

in this document.

Contents

Contents and storage

Table1ProQuest™ Two-Hybrid System (Cat. No. PQ10001-01)

Component[1] Storage

ProQuest™ Vectors -80°C

ProQuest™ Control Vectors -20°C

LR Clonase™ II -20°C

[1] All components are shipped on dry ice.

Shipping and storage

The ProQuest™ Two-Hybrid System is shipped as described below. Upon receipt, store each item as

detailed below.

Box Component Shipping Storage

1 ProQuest™ Vectors Dry ice -80℃

2 ProQuest™ Control Vectors Dry ice -20℃

3 LR Clonase™ II Dry ice -20℃

8ProQuest™ Two-Hybrid System User Guide

ProQuest™ Two-Hybrid system reagents

The following reagents are included with the ProQuest™ Two-Hybrid System Reagents.

ProQuest™ Vectors Box (Store the reagents at -80℃)

Reagent Composition Amount

pDEST™22 Liquid in TE Buer, pH 8.0 6µg

pDEST™32 Liquid in TE Buer, pH 8.0 6µg

MaV203 glycerol stock YPAD + 20% glycerol 0.5mL

ProQuest™ Control Vectors Box (Store the reagents at -20℃)

Reagent Composition Amount

pEXP™-AD502 Liquid in TE Buer, pH 8.0 1µg

pEXP™32/Krev1 Liquid in TE Buer, pH 8.0 10µg

pEXP™22/RalGDS-wt Liquid in TE Buer, pH 8.0 10µg

pEXP™22/RalGDS-m1 Liquid in TE Buer, pH 8.0 10µg

pEXP™22/RalGDS-m2 Liquid in TE Buer, pH 8.0 10µg

LR Clonase™ II Box

(Store at -20℃ for up to 6 months. For long-term storage, store at -80℃.)

Reagent Composition Amount

Gateway™ LR Clonase™ II Enzyme Mix Proprietary 40µL

Proteinase K Solution Solution 2µg/µL in:

10 mM Tris-HCl, pH 7.5

20 mM CaCl2

50% glycerol

40µL

pENTR™-gus Positive Control 50ng/µL in TE Buer, pH 8.0 20µL

Genotype MaV203

The genotype of MaV203 is as follows:

MaV203 (MATα, leu2-3,112, trp1-901, his3Δ200, ade2-101, gal4Δ, gal80Δ, SPAL10::URA3, GAL1::lacZ,

HIS3UAS GAL1::HIS3@LYS2, can1R, cyh2R)

Chapter1Product information

Contents 1

ProQuest™ Two-Hybrid System User Guide 9

Accessory products

Introduction

The products listed in this section may be used with the ProQuest™ Two-Hybrid System. For more

information, see (thermoﬁsher.com).

MaV203 competent cells

We provide a glycerol stock of the MaV203 yeast strain. A protocol is provided to perform small-scale

transformations using this stock. To limit your workload, purchase competent MaV203cells, subclone

scale. Alternatively, prepare competent cells using the S. c. EasyComp™ Kit, which can be frozen for

later use. For large-scale applications, such as a forward two-hybrid library screen, we recommend

obtaining MaV203 Competent Cells, Library Scale to get the highest transformation eciency and to

limit your workload.

Item Amount Catalog no.

MaV203 Competent Cells, Library Scale 2 x 0.55ml 11281011

MaV203 Competent Cells, Subclone Scale 4 x 0.10ml 11445012

S. c. EasyComp™ Kit 1 kit K505001

Note: For your convenience, we have added a protocol in the (Appendix C, “Supplementary protocols”)

to make your own large-scale competent cells using MaV203 cells provided with the kit.

Accessory products

Some of the reagents supplied in the ProQuest™ Two-Hybrid System and as well as other products

suitable for use with the kit are available separately from thermoﬁsher.com. Ordering information is

provided below.

Item Amount Catalog no.

PureLink™ HQ Mini Plasmid DNA Puriﬁcation Kit 100 preps K210001

PureLink™ HiPure Plasmid Miniprep Kit 25 preps

100 preps

K210002

K210003

PureLink™ HiPure Plasmid Midiprep Kit 25 preps

50 preps

K210004

K210005

5‑bromo-4‑chloro-3‑indolyl-D-galactopyranoside (X-Gal), 100mg

1g

15520034

15520018

Denatured Sheared Salmon Sperm DNA 5 x 1mL 15632011

OneShot™ TOP10 Electrocomp™ E. coli 10 reactions

20 reactions

C404050

C404052

Chapter1Product information

Accessory products

10 ProQuest™ Two-Hybrid System User Guide

(continued)

Item Amount Catalog no.

Platinum™ PCR SuperMix HiFi 100 reactions 12532016

Platinum™ PCR SuperMix 100 reactions 11306016

Platinum™ Taq DNA Polymerase 100 reactions 10966018

S.N.A.P.™ Gel Puriﬁcation Kit 25 reactions K199925

2.5mM dNTP Mix 1mL R72501

Gateway™ LR Clonase™ II Enzyme Mix 20 reactions

100 reactions

11791020

11791100

Gateway™ BP Clonase™ II Enzyme Mix 20 reactions

100 reactions

11789020

11789100

Proteinase K Solution 100mg

25530015

25530031

pCR™8/GW/TOPO™ TA Cloning™ Kit 20 reactions K250020

Gentamicin Reagent Solution (10mg/mL), liquid 10mL

10 x 10mL

15710064

15710072

SOC Medium 10 x 10mL 15544034

LB Agar, powder (Lennox L Agar) 500g

2.5kg

22700025

22700041

LB Broth Base, powder (Lennox L Broth Base) 500g

2.5kg

12780052

12780029

3-Aminotriazole

For selection of HIS+ transformants in S. cerevisiae, you will need to obtain 3‑aminotriazole, which is

available from Millipore Sigma, St. Louis, MO, (Catalog No. A8056).

Zymolyase™

For isolation of yeast DNA you need Zymolyase™ (1.5 U/µL), which is available from G-Biosciences,

St.Louis, MO (Catalog no. 786-036).

Cycloheximide

For Plasmid shuing (see “Plasmid shuing” on page94), you will need to obtain cycloheximide,

which is available from Millipore Sigma, St. Louis, MO (Catalog No. C1988).

Chapter1Product information

Accessory products 1

ProQuest™ Two-Hybrid System User Guide 11

Yeast media

For yeast selective media, recipes are provided in AppendixB, “Recipes”.

Chapter1Product information

Accessory products

12 ProQuest™ Two-Hybrid System User Guide

General introduction

Overview

ProQuest™ Two-Hybrid System

The ProQuest™ Two-Hybrid System is a genetic method for detecting interactions between proteins in

vivo in the yeast Saccharomyces cerevisiae. The ProQuest™ Two-Hybrid System draws on modiﬁcations

by Chevray & Nathans and incorporates Gateway™ Technology.

Supported applications

The ProQuest™ Two-Hybrid System supports three types of applications:

•Verifying an interaction between two known proteins or protein domains for which there is a

prior reason to expect an interaction (testing two-hybrid interactions); see Chapter 3, “Verifying

interaction”.

•Screening a library for novel proteins that speciﬁcally interact with a known bait (forward two-hybrid

library screen); see Chapter 4, “Forward Two-Hybrid library screen”.

Advantages of the ProQuest™ Two-Hybrid system

The ProQuest™ Two-Hybrid System is a system designed to enable detection of protein-protein

interactions and has been modiﬁed to decrease false positives. The primary modiﬁcations include:

•Uses low-copy-number (ARS/CEN) vectors to control over-expression and increase reproducibility

•Contains three dierent reporter genes with independent promoters to rapidly weed out false

positives

•Uses a reporter gene (URA3) that allows both positive and negative selection

•An extended panel of yeast control vectors to aid in setting up the experiments and evaluate results

•Incorporation of the Gateway™ Technology to allow rapid and easy generation of bait and prey

constructs, and to facilitate down-stream applications

System components

The ProQuest™ Two-Hybrid System includes:

•Yeast expression vectors pDEST™22, pDEST™32, and pEXP™-AD502 for generation of GAL4 DNA

Binding Domain (GAL4 DBD) and GAL4 Activation Domain (GAL4 AD) fusion proteins

•Reagents for production of the expression clones containing GAL4 DBD and GAL4 AD fusion

proteins

•A glycerol stock of yeast strain MaV203, which is the two-hybrid yeast strain used

•Positive and negative controls for the two-hybrid assay

ProQuest™ Two-Hybrid System User Guide 13

Gateway™ Technology

All yeast expression vectors in the ProQuest™ Two-Hybrid System are Gateway™-adapted to allow rapid

and easy generation of bait and prey constructs, and to facilitate down-stream applications.

The Gateway™ Technology is a universal cloning method that takes advantage of the site-speciﬁc

recombination properties of bacteriophage lambda to provide a rapid and highly ecient way to move

your DNA sequence of interest into multiple vector systems.

For a brief description of the Gateway™ Technology, see the Appendix, AppendixA, “Gateway™

recombination reactions”

Purpose of this manual

This manual provides the following information:

•An overview of the two-hybrid technology (“General description of the Two-Hybrid system” on

page15)

•Instructions to make your bait and prey plasmid (“Generating bait and prey plasmids” on page31)

•Guidelines for testing the interaction between two proteins (“Recommended controls” on page40)

•Guidelines for choosing the library you want to screen (“Choosing Two-Hybrid library” on page49)

•Procedures to perform forward two-hybrid library screens (“Screening forward Two-Hybrid library”

on page55)

Important

The ProQuest™ Two-Hybrid System is designed to help you perform your two-hybrid analysis. The

system has been designed to help you perform your experiment in the simplest, most direct fashion,

but use of the system assumes that users are familiar with manipulating yeast and cloning.

Chapter2General introduction

Overview

14 ProQuest™ Two-Hybrid System User Guide

General description of the Two-Hybrid system

Two hybrid proteins

Two-hybrid or interaction trap systems exploit the fact that transcription factors are comprised of two

domains, a DNA binding domain (DBD) and an activation domain (AD). Two separate hybrid proteins

are constructed in two-hybrid screens. The ﬁrst hybrid protein is the DBD/protein X fusion known as

the “bait”, while the second hybrid protein is the AD/protein Y fusion known as the “prey”. These two

hybrids are encoded on separate yeast expression plasmids, with independent selectable markers.

Figure1Bait

Figure2Prey

Chapter2General introduction

General description of the Two-Hybrid system 2

ProQuest™ Two-Hybrid System User Guide 15

Reporters under control of UAS

The yeast strain employed contains reporter genes, such as lacZ or auxotrophic markers such as

HIS3 or URA3. The regulatory regions of these reporters have been engineered to contain the DNA

binding sites (operator sequences) for the DBD/protein X fusion (bait). These operator sequences act as

upstream activating sequences (UAS) in yeast.

Note: Yeast two-hybrid strains have been speciﬁcally modiﬁed to contain these reporter genes. wt

yeast strains cannot be used!

Interaction drives expression of reporters

The yeast strain used is transformed with the expression plasmids encoding the bait and prey. If

protein X interacts with protein Y in the nucleus, this will bring the activation domain together with the

DNA-binding domain to reconstitute transcriptional activation and result in expression of the reporter

genes.

X and Y do not interact - no Reporter Gene Expression

Chapter2General introduction

General description of the Two-Hybrid system

16 ProQuest™ Two-Hybrid System User Guide

X and Y interact - Reporter Gene Expressed!

Evaluating reporter gene expression

There are two main ways to check for positive interactions in yeast strains containing reporter genes:

•Positive interactions are detected by selecting on plates lacking the auxotrophic marker, such as

Histidine or Uracil. Yeast cells containing plasmids that express interacting bait and prey proteins

will grow and form colonies.

•Positive interactions are detected by assaying for enzyme activity, such as colorimetric assays for

β-galactosidase activity. This is used to reduce false positives after selection for auxotrophs, or to

measure interaction strength quantitatively.

Screening Two-Hybrid libraries

Two-hybrid libraries (i.e. prey libraries) consist of a collection of expression plasmids in which an

Activation Domain is fused to individual cDNAs. Screening prey libraries will detect prey proteins that

interact with the bait protein of interest. To perform the screen, transform the library and bait expressing

plasmids into yeast. Cells containing a prey that interacts with the bait will form colonies on selective

plates. Secondary screens, such as for β-galactosidase expression, will conﬁrm the interaction.

False positives

Early two-hybrid systems suered from false positives - candidate proteins identiﬁed as interacting but

which do not truly interact or are biologically irrelevant. False positives can result from:

•Proteins containing regions with surfaces having low anities for many dierent proteins, (e.g.,

large hydrophobic surfaces)

•Proteins that normally interact with a large number of proteins (e.g., heat shock proteins)

•Proteins containing regions functioning as activation domains

•Proteins aecting chromatin structure

•Proteins having low or nonspeciﬁc anities for the promoter regions (or proteins bound there) that

drive the expression of reporter genes

Limiting the false positives is essential in successful two-hybrid experiments.

Chapter2General introduction

General description of the Two-Hybrid system 2

ProQuest™ Two-Hybrid System User Guide 17

Advanced™ Two-Hybrid systems

The ProQuest™ Two-Hybrid System has been extensively improved to limit false positives, as well as to

allow performance of more advanced applications. For details about the ProQuest™ Two-Hybrid System

and strategies to limit false positives, see “Reducing false positives” on page21.

Chapter2General introduction

General description of the Two-Hybrid system

18 ProQuest™ Two-Hybrid System User Guide

ProQuest™ Two-Hybrid system

Verifying Two-Hybrid interaction

Using the ProQuest™ Two-Hybrid System to verify an interaction between two known proteins, you will

perform the following steps:

1. Construct bait plasmid

2. Construct prey plasmid

3. Transform yeast cells with bait and prey plasmid

4. Test reporter activity

Forward Two-Hybrid library screen

Using the ProQuest™ Two-Hybrid System for a forward two-hybrid library screen, you will perform the

following steps:

1. Construct and test bait plasmid

2. Construct or obtain two-hybrid library

3. Transform yeast cells with bait plasmid and library

4. Select for reporter activity by growth on auxotrophic plates

5. Conﬁrm interaction of positive prey plasmids

Note: The ProQuest™ Two-Hybrid System comes with a positive interaction control. However, a

bait-speciﬁc positive interaction control may be an additional useful tool in testing your experiment.

Construct a prey plasmid with a known interactor of the bait protein to use as a bait-speciﬁc positive

control.

Cloning vectors

The ProQuest™ Two-Hybrid System includes these yeast expression vectors:

•pDEST™32 for generation of the bait plasmid

•pDEST™22 for construction of the prey plasmid, or for generation of a two-hybrid library by

Gateway™ recombination

•pEXP™-AD502 for generation of a two-hybrid library by restriction cloning

Chapter2General introduction

ProQuest™ Two-Hybrid system 2

ProQuest™ Two-Hybrid System User Guide 19

Two-Hybrid control vectors

The ProQuest™ Two-Hybrid System includes four two-hybrid control plasmids based on the interaction

of Krev1 (a.k.a. Rap1A; a member of the Ras family of GTP binding proteins) with RalGDS (the Ral

guanine nucleotide dissociator stimulator protein. The RalGDS mutants RalGDS-m1 and RalGDS-m2

aect the interaction with Krev1 and were generated using the SureFrame™ Allele Library Construction

Kit. The properties of these plasmids are summarized below.

Control plasmid Backbone Insert Mutant Role Interaction with

pEXP™32/Krev1

pEXP™32/ Krev1 pDEST™32 full-length rat Krev1 wt Bait not applicable

pEXP™22/ RalGDS-

pDEST™22 ras association domain of

RalGDS, wt

wt Prey strong

pEXP™22/ RalGDS-

pDEST™22 ras association domain of

RalGDS, m1

I77T[1] Prey weak

pEXP™22/ RalGDS-

pDEST™22 ras association domain of

RalGDS, m2

L65P1Prey not detectable

[1] Amino acid 65 and amino acid 77in the insert correspond to amino acid 829 and amino acid 841in the full-length RalGDS sequence,

respectively.

The vectors pDEST™32 and pDEST™22 are suitable as negative two-hybrid controls.

•pDEST™32 as a negative control for bait plasmid

•pDEST™22 as a negative control for prey plasmid

Chapter2General introduction

ProQuest™ Two-Hybrid system

20 ProQuest™ Two-Hybrid System User Guide

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